title: In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils.
abstract: Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.
authors.affiliation: Department of Infection, Immunity and Biochemistry, Cardiff University School of Medicine, Tenovus Bldg., Heath Park, Cardiff, CF14 4XN, UK.
authors.forename: Jacqueline U
authors.initials: JU
authors.lastname: McDonald
authors.forename: Andrea
authors.initials: A
authors.lastname: Cortini
authors.forename: Marcela
authors.initials: M
authors.lastname: Rosas
authors.forename: Liliane
authors.initials: L
authors.lastname: Fossati-Jimack
authors.forename: Guang Sheng
authors.initials: GS
authors.lastname: Ling
authors.forename: Kimberley J
authors.initials: KJ
authors.lastname: Lewis
authors.forename: Sharon
authors.initials: S
authors.lastname: Dewitt
authors.forename: Kate
authors.initials: K
authors.lastname: Liddiard
authors.forename: Gordon D
authors.initials: GD
authors.lastname: Brown
authors.forename: Simon A
authors.initials: SA
authors.lastname: Jones
authors.forename: Maurice B
authors.initials: MB
authors.lastname: Hallett
authors.forename: Marina
authors.initials: M
authors.lastname: Botto
authors.forename: Philip R
authors.initials: PR
authors.lastname: Taylor
mesh_terms: D023401:Animal Use Alternatives; D000818:Animals; D039481:CD11b Antigen / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D005786:Gene Expression Regulation / Q000502:physiology; D005818:Genetic Engineering / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009154:Mutation; D009504:Neutrophils / Q000166:cytology* / Q000502:physiology*; D014161:Transduction, Genetic / Q000379:methods; D015003:Yeasts
chemical_list: D039481:CD11b Antigen; D018398:Homeodomain Proteins; C095822:Hoxb8 protein, mouse